It will be clearly understood that, if a prior art publication is referred to herein, this reference does not constitute an admission that the publication forms part of the common general knowledge in the art in Australia or in any other country.
The discussion below especially relates to the protection of plants against insect populations, and especially the control of insect populations that eat plants. However, for the avoidance of doubt, the present invention is not limited to protection of plants or the control of such insect populations. For example, many of the issues with chemical agents for controlling insects discussed below are also of concern for non-agricultural insect control.
Two of the most common strategies for the control of insect pests on plants include breeding for disease resistance and chemical control. Chemical control in particular is frequently used to control insect populations, but chemical agents are often expensive. Furthermore, there are safety concerns relating to the potential impact of chemical agents on the environment and the use of chemical agents on, for example, fruits and vegetables to be consumed by people. These last issues are factors that have contributed to the growth of the market for organic fruit and vegetables.
A further critical factor is that insects can and have developed resistance to traditional chemical agent insecticides. For example, in Australia field populations of insecticide-resistant Helicoverpa armigera (Cotton bollworm) have developed, and these resistant sub-types represent a significant pest in Australia's northern grain region (which encompasses all of Queensland's production areas). Crops affected by H. armigera include canola, chickpea, cotton, lucerne, maize, millets and panicums, mungbeans, peanuts, sorghum, soybeans, sunflowers and winter cereals including wheat, barley, oats, canary and triticale. H. armigera is also a major agricultural pest endemic to Europe, Asia and Africa. It is highly polyphagous and has been shown to feed on greater than 180 species of plant in 47 families. H. armigera has been estimated to cause US$2 billion in crop damage annually, despite the expenditure of US$500 million worth of pesticides for its control. However, H. armigera is just one species in the order of Lepidoptera, which is the second largest insect order comprised of moths and butterflies. The larval stage of moths cause major damage to many economically valuable crops including cotton, tobacco, tomato, corn, sorghum, lucerne, sunflower, pulses and wheat.
One effort to reduce the use of chemical insecticides on plants has involved the development of transgenic plants. For example, over the last two decades there has been an increase in the adoption of transgenic plants expressing insecticidal proteins encoded by genes from the bacterium Bacillus thuringiensis (Bt). However, the availability of suitable plants is limited, and sub-types of H. armigera, for example, have developed resistance to both single and dual-toxin forms of Bt cotton. Furthermore, large-scale application of transgenic plants has encountered resistance from the public and from regulatory agencies, and the cost, laboriousness and time needed to develop transgenic plants makes this an unattractive option.
RNAi, or RNA interference (also known as RNA silencing), has been used for genetic research in insects and it has promise in entomology as it provides target-specific silencing of gene expression. However, there are many difficulties in using RNAi to control the impact of insect pests on plants.
The RNAi mechanism involves first introducing double stranded RNA (dsRNA) into the insect haemocoel (for example via ingestion/injection/soaking), after which the dsRNA proceeds to enter the insect cells via endocytosis or via transmembrane proteins. Next, dsRNA is digested within the insect cells by a ribonuclease III (RNaseIII) enzyme known as Dicer into short interfering RNAs (siRNAs) of 20-25 nucleotides in length. The siRNAs are then unwound and one strand (the guide strand) is loaded into the RNA induced silencing complex (RISC). Lastly, the RISC locates and binds to messenger RNAs (mRNAs) containing sequences complementary to the guide strand, resulting in degradation of mRNA of the target gene, finally leading to the death of the cell.
A major difficulty in using the RNAi mechanism is how to effectively deliver dsRNA to insects. In particular, most laboratory studies of RNAi in insects have utilised microinjection of in vitro synthesised dsRNAs into embryos or the hemocoel. Clearly, however, microinjection as a delivery mechanism is not feasible for large-scale plant protection. Another strategy is for insects to ingest the dsRNA by providing it mixed in the diet, but this means that the dsRNA enters the insect alimentary canal, and especially the midgut. The environment of the midgut is hostile to dsRNA, due to the pH of the midgut and the presence of gut nucleases. Nucleases capable of degrading dsRNA may also be present in the saliva of insects (as recently reported for the tarnished plant bug Lygus lineolaris by Allen and Walker (2012) Saliva of Lygus lineolaris digests double stranded ribonucleic acids. Journal of Insect Physiology, 58, 391-396). Furthermore, research indicates that systemic RNAi responses may be more robust in less derived insect species, but some more derived Dipteran and Lepidopteran species may be refractory to systemic RNAi.
Another difficulty is how to provide the dsRNA to insects for ingestion. dsRNA is vulnerable to nucleases in the environment, and also to ultraviolet light, specifically if it has to be provided as a topical application on the plant surface. For example, in one study dsRNA topically applied to a plant for protection against viruses could not protect the plants 7 days post application. Furthermore, when dsRNA was applied 24 hours after viral infection, the dsRNA was not able to protect the plants against the virus (Tenllado, F. & J. R. Diaz-Ruiz, (2001) Double-stranded RNA-mediated interference with plant virus infection. Journal Virology 75: 12288-12297). In another study, when dsRNA was added to insect foods, the dsRNA levels in different diets dropped by 14% in the solid foods for beetles and moths and by 32% in the aphid fluid diet after 3 days, and by 31% and 56% after 6 days, respectively (Whyard et al (2009) Ingested double stranded RNAs can act as species-specific insecticides. Insect Biochemistry and Molecular Biology, 39, 824-832).
Major challenges for application of RNAi to insects (after effective gene targets have been identified) are the stability of dsRNA prior to administration to the insect, and the delivery of dsRNA to the insect.
Consequently, there is a need to provide an effective alternative approach which allows for targeting of insect populations or an approach which at least partially overcomes at least one of the abovementioned disadvantages or which provides the consumer with a useful or commercial choice.